We also encountered this problem when we set the procedure up in our lab.
The reason why we couldn't successfully reprobe was probably due to the
membrane being stored for 1 or more days soaked in the chemiluminescent
substrate after exposing to film (prolonged exposure to the high pH of
the substrate buffer was perhaps causing degradation of the blotted RNA).
We overcame the problem by stripping the membrane immediately after the
exposures were completed and either sealing it wet (soaked in 2XSSC) in a
clean polythene bag in the fridge or prehybridising it prior to
reprobing. The stripping procedure we use is as follows:
DEPC-water for 5 min
Boiled 0.1% SDS for 10 min
100 mM maleic acid, 150 mM NaCl, 0.3% Tween 20, pH 7.5 (i.e. Boehringer
1X Washing buffer) for 5 min
2X SSC for 5 min
Repeat last step.
(All steps performed on a rocking platform.)
Hope this is useful.
Gary Moore
--
The opinions expressed in this communication are my own,
and do not necessarily reflect those of my employer.